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DPDL1E Vaccine-Induced Cytotoxic T Lymphocyte (CTL) Response
Because the vaccine induced a Th1-biased type of immunity, we examined the PD-L1-specific CTL response using a lactate dehydro-
Figure 1. Design of the DPDL1E Vaccine Antigen and Its Recombinant Preparation
(A) Schematic representation of the DPDL1E antigen linear structure. (B) Structure model of DPDL1E. (C) SDS-PAGE analysis of DPDL1E antigen expression and purification. Lane 1: induced whole-cell lysate of DPDL1E with the GST tag. Lane 2: induced supernatant of DPDL1E with the GST tag. Lane 3: purified recombinant protein of DPDL1E with the GST tag. Lane 4: product mixture of GST-DPDL1E after PSP digestion. Lane 5: purified DPDL1E.
genase (LDH) release assay. Lymphocytes from DPDL1E-immunized mice were used as effector cells, and PD-L1+ B16-F10 FF-MAS were used as target cells. As expected, a significantly high level of cyto-toxic activity was observed in DPDL1E-immunized mice compared with DTT-immunized mice (p < 0.01) (Figure 3A). Moreover, splenocytes from DPDL1E-immunized mice had a higher stimula-tion index than those from DTT-immunized mice with His-PD-L1 as a stimulator in vitro (p < 0.05), indicating that PD-L1-specific memory
T cells had developed (Figure 3B). We measured the cytokine levels in the culture supernatants by ELISA. Compared with the DTT con-trol group, the concentrations of interleukin-2 (IL-2), IFN-g, and tumor necrosis factor alpha (TNF-a) were increased (153.25 pg/mL versus 975.25 pg/mL, 147.5 pg/mL versus 936.25 pg/mL, and 26.32 pg/mL versus 111.25 pg/mL, respectively). We further analyzed PD-L1-induced T cell proliferation in vitro by carboxyfluorescein succinimidyl ester (CFSE) profiling (Figure 3C) and found that PD-L1-specific CD8+ T cells and CD4+ T cells were present in immu-nized mice splenocytes (Figure 3D), demonstrating that DPDL1E vaccination can elicit PD-L1-specific cellular immune responses.
DPDL1E Inhibits Tumor Growth in a Preventive Mouse Model
To evaluate the efficacy of the vaccine in tumor growth control, we chal-lenged DPDL1E-immunized mice with B16-F10 cells (Figure 4A) and monitored tumor development in comparison with DTT- or PBS-treated mice. We found that the DPDL1E vaccination significantly in-hibited tumor growth, and the median survival was increased from 16 days (PBS group) and 15 days (DTT group) to 23 days (DPDL1E group). Notably, 25% of DPDL1E-vaccinated mice had no tumor growth, and their survival was prolonged beyond 60 days (Figures 4B, 4C, and S1A). We also noted that the tumor volumes of DPDL1E-immunized mice on day 19 after tumor challenge were correlated with the PD-L1-specific antibodies titers on day 7 after the third immu-nization (R2 = 0.6425) (Figure 4D), which indicated that anti-PD-L1 an-tibodies are the major contributors to tumor control. In parallel exper-iments, we found that the DPDL1E vaccine also inhibits tumor growth in the CT26 colon cancer mouse model (Figure S2).
Because B16-F10 is a metastatic cell line, we asked whether DPDL1E vaccination could inhibit metastasis to the lungs. We applied the
Molecular Therapy: Oncolytics
Figure 2. Antibody Responses Induced by DPDL1E Vaccination in C57BL/6 and BALB/c Mice
(A) Mice (n = 8) were immunized with DPDL1E three times at 2-week intervals. One week after the third immunization, the antibody titers were measured by ELISA using His-tagged PDL1 recombinant protein as a coating antigen. DTT-immunized serum was used as a negative control. (B) The levels of PD-L1- and DTT-specific antibody subclasses induced by DPDL1E vaccination. Sera were isolated from C57BL/6 and BALB/C mice immunized with the DPDL1E vaccine. The levels of the indicated antibody subclasses were measured by ELISA. (C) The sera from DPDL1E-immunized mice inhibited binding of PD-L1 to PD-1. PD-L1 mAb at 20 mg/mL was used as a positive control, and sera from DTT-immunized mice and PBS were used as a negative control and blank control, respectively. (D) The inhibition efficiency of sera at different concentrations was tested and compared with the control group. (E) A standard curve was created (relative inhibition versus concentration of PD-L1 mAb) to calculate the effective anti-PD-L1 concentration in vitro. ****p < 0.0001.